Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Thirteen genes have now been described that are mutated to cause FA, and three are discovered by my group. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in that pathway is a modification of an FA protein, FANCD2. The modification, monoubiquitylation, results in redistribution of FANCD2 to specific spots in the nucleus where BRACA1 also localizes. Five other FA proteins (FANCA, -C, -E, -F, and -G) have been found to interact with each other to form a multiprotein nuclear complex, the FA core complex. This complex functions upstream in the pathway and is required for FANCD2 monoubiquitylation. We have purified the FA protein core complex and found that it contains four new components in addition to the five known FA proteins. One new component of this complex, termed PHF9, possesses ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitylation in vivo. PHF9 is defective in a cell line derived from a Fanconi anemia patient, and therefore represents a novel Fanconi anemia gene (FANCL). Our data suggest that PHF9 plays a crucial role in the Fanconi anemia pathway as the catalytic subunit required for FANCD2 monoubiquitylation. The discovery of PHF9/FANCL might provide a potential target for new therapeutic modalities. We then showed that the 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). The significance of this study can be summarized as follows. First, our study identifies the true FANCB gene that had eluded identification for more than 10 years. Before our paper, the identity of the FANCB gene was controversial, and has been suggested to be BRCA2. Our paper settled this issue for the field. Second, we find that FANCB is localized on the X-chromosome and subject to X-inactivation. This finding has changed the prevalent view that Fanconi anemia is a uniquely autosomally-inherited disease. Our paper thus has important clinical implication for diagnosis and genetic counseling for FA families. For example, the female carriers of FANCB mutation will have 50 percent of risk to conceive an affected son or a carrier daughter. These carriers should be identified and given proper counseling for risk. Third, all other genes that maintain genome stability are localized on autosomes and present in two copies. In contrast, FANCB is X-linked and present in only one active copy. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability, because it will only take one mutation to inactivate FANCB, compared to two mutations required to inactivate other FA genes. Our study suggests that FANCB may be mutated in cancer patients who do not have Fanconi anemia. We demonstrated that FAAP250 is mutated in FA patients of a new complementation group, FA-M. The gene encoding the FAAP250 protein was renamed FANCM. The importance of the FANCM findings is that it has a conserved helicase domain and a DNA-translocase activity. A companion paper identified another FA protein, FANCJ, as BACH1/BRIP1, a known DNA helicase. The discovery of two FA proteins with helicase domains or activities suggests a mechanism of direct participation in DNA repair by the FA proteins. FANCM may have at least three important roles in the FA DNA damage response pathway. First, FANCM may have a structural role to allow assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM may translocates and remodel various DNA structures, which may be important for subsequent DNA repair. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. We have recently identified another component of the FA core complex, FAAP100, and shown that this protein is required for stability and a key function of the complex--FANCD2 monoubiquitination. Thus, all nine components of the core complex are essential for the ubiquitination reaction, suggesting that the entire complex is a machine that works concertedly to monoubiquitinate FANCD2. We also found that deficiency in FAAP100 generated by siRNA depletion or gene knockout results in cellular phenotypes that are hallmark features of FA cells. Therefore, FAAP100 should be an essential component of the FA-BRCA network, and its defects in human should also cause FA. More recently, we have identified a new component of the FA core complex, termed FAAP24. FAAP24 contains an ERCC4-like endonuclease domain, and forms a heterodimer with FANCM. We find that FAAP24 can recognize structured DNA that mimics intermediates generated during DNA replication. Moreover, it can targets FANCM to such structures. Cells depleted of FAAP24 show phenotypes that are characteristics of FA cells. Our results demonstrate that FAAP24 is a new essential component of the FA core complex, and its defect could cause FA. We also collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. We demonstrated that FANCM possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA crosslinking drug, mitomycin C (MMC), but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP- dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair. We identified two new components in the FA core complex, FAAP16 and FAAP10. Both FAAP16 and FAAP10 have a DNA binding domain. We found that these two proteins form a stable subcomplex, which binds dsDNA (though not ssDNA). Importantly, while the FANCM helicase domain binds little to dsDNA, addition of the FAAP16-10 complex strongly enhanced the DNA binding activity of FANCM. In collaboration with Contantinous lab (Lausanne University), we found that the FAAP16-10 complex stimulates the ATP-dependent replication fork reversal activity of FANCM, indicating that it could be an important cofactor for FANCM in DNA repair. Importantly, depletion of either FAAP16 or FAAP10 results in reduced monoubiquitination of FANCD2 and FANCI, cellular hypersensitivity to DNA damaging drugs, and chromosomal breakage, indicating that FAAP16-10 plays an important role in the FA pathway. We knocked out the FAAP16 gene in chicken DT40 cells. The FAAP16 mutant cells display several features that resemble FANCM mutant cells, including reduced FANCD2 monoubiquitination and increased level of SCE. The data provide genetic evidence that FAAP16 is important for activation of the FA pathway in chicken DT40 cells. We found that another FA protein, FANCJ, becomes hyperphosphorylated in response to DNA damage. We are investigating if this phosphorylation regulates activity of FANCJ in DNA repair. We are continuing to identify and characterize additional components of the FA complex. The eventual goal is to find new targets for drug interventions.